A multidisciplinary group at UCLA (Andre Nel, Jeffrey Zink, Tian Xia, Ning Li) in collaboration with Dr. Vince Castranova at NIOSH and Dr. Lutz Mddler at Bremen University (Germany), aim to establish a mouse inhalation toxicology model to screen combinatorial nanomaterials libraries that are linked to mechanistic injury pathways in tissue culture cells. This research will address the lack of reproducible screening protocols for assessment of nanomaterial (NM) safety. We hypothesize that two combinatorial libraries, which have been designed to (i) adjust the toxic effects of ZnO nanoparticles by changing Zn++ release through iron doping, (ii) adjust the cytotoxicity of cationic mesoporous silica nanoparticles (MSNP) by scaling back the surface cationic density, will be useful to establish a link between in vitro toxicology in human bronchial epithelium (NHBE) and myeloid dendritic cells (DC) and non-allergic and allergic pulmonary inflammation in mice. We posit that the link between non-allergic airway inflammation and cytotoxic injury will be explicable by nanoparticle properties that lead to oxidant injury, shedding of toxic metal ions, and ability to trigger mitochondrial injury. In contrast, the linkage of the material properties to allergic airway inflammation is likely to impact the generation of "danger signals" to DC, which allow them to initiate an immunostimulatory pathway that promotes allergic inflammation. To achieve our long-term goal of developing a predictive inhalation toxicology model, we propose in Aim 1 to characterize the bio-physicochemical properties of NM libraries that are expected to induce differential cytotoxic and pro-inflammatory effects in NHBE and bone marrow-derived DC. In vitro toxicity screening will be carried out by the high content screening (HCS) facility in the California Nano Systems Institute at UCLA as well as cytokine measurements by ELISA. We will also assess ROS production. The NM properties that will be assessed include particle size, size distribution, dispersibility, zeta-potential, TEM, XRD and BET analysis. HCS is carried out with a cocktail of dyes that reveal cellular membrane leakage, DNA damage, mitochondrial depolarization and intracellular Ca2+ flux. The particle characterization and in vitro screening will be carried out at the beginning of year 1 and when new batches are synthesized. Aim 2 will use the libraries to establish standardized protocols for allergic and non-allergic pulmonary inflammation in mice by an inhalation-aspiration approach. The protocol for non-eosinophilic inflammation (Castranova) will measure markers of inflammation, damage, and oxidant stress in the BAL along with lung histology for period of 1 day up to 2 months post-exposure. The allergic sensitization model, which relies on particle and OVA aspiration from the nose of anesthesized animals, will assess OVA-specific IgE levels in parallel with BAL differential cell counts and lung histology. These studies will be carried out in phases through years 1-2. We expect to develop a novel and predictive inhalation toxicology paradigm as a component of the consortium activities to establish in vitro and in vivo study protocols for NM safety screening. PUBLIC HEALTH RELEVANCE: This project addresses the design of cellular and animal screening procedures that can be used to assess the potential danger of engineered nanomaterials that are currently being introduced into the marketplace. We delineate a unique approach in which we use in-house synthesized nanoparticles to develop a predictive toxicological model, meaning that we will develop test protocols in tissue culture cells to obtain basic information about the nanoparticle properties that may render them dangerous and then test those ideas out in a mouse model that may reflect what could happen in the lung when those particles are inhaled. The goal of this project is to develop reliable and reproducible protocols that can be used by academic centers, industry and government agencies as a screen for nanomaterial safety.